RESUMEN
Bicyclic peptides, marinostatins, are protease inhibitors derived from the marine bacterium Algicola sagamiensis. The biosynthetic gene cluster of marinostatin was previously identified, although no heterologous production was reported. In this report, the biosynthetic gene cluster of marinostatin (mstA and mstB) was cloned into the expression vector pET-41a(+). As a result of the coexpression experiment, a new analogous peptide named marinostatin E was successfully produced using Escherichia coli BL21(DE3). The structure of marinostatin E was determined by a combination of chemical treatments and tandem mass spectrometry experiments. Marinostatin E exhibited inhibitory activities against chymotrypsin and subtilisin with an IC50 of 4.0 and 39.6 µm, respectively.
Asunto(s)
Ingeniería Genética , Péptidos Cíclicos/biosíntesis , Inhibidores de Proteasas/metabolismo , Gammaproteobacteria/genética , Familia de Multigenes/genética , Péptidos Cíclicos/química , Péptidos Cíclicos/genética , Inhibidores de Proteasas/químicaRESUMEN
Recently, ω-ester-containing peptides (OEPs) were indicated to be a class of ribosomally synthesized and post-translationally modified peptides. Based on genome mining, new biosynthetic gene cluster of OEPs was found in the genome sequence of actinobacterium Streptomyces prunicolor. The biosynthetic gene cluster contained just two genes including precursor peptide (pruA) and ATP-grasp ligase (pruB) coding genes. Heterologous co-expression of the two genes was accomplished using expression vector pET-41a(+) in Escherichia coli. As a result, new OEP named prunipeptin was produced by this system. By site-directed mutagenesis experiment, a variant peptide prunipeptin 15HW was obtained. The bridging pattern of prunipeptin 15HW was determined by combination of chemical cleavage and MS experiments. Prunipeptin 15HW possessed bicyclic structure with an ester bond and an isopeptide bond. The ATP-grasp ligase PruB was indicated to catalyze the two different intramolecular bonds.
Asunto(s)
Escherichia coli/metabolismo , Familia de Multigenes , Péptidos/metabolismo , Streptomyces/genética , Vías Biosintéticas , Escherichia coli/genética , Expresión GénicaRESUMEN
Lasso peptides produced by bacteria have a very unique cyclic structure ("lasso" structure) and are resistant to protease. To date, a number of lasso peptides have been isolated from proteobacteria and actinobacteria. Many lasso peptides exhibit various biological activities, such as antibacterial activity, and are expected to have various applications. Based on study of genome mining, large numbers of biosynthetic gene cluster of lasso peptides are revealed to distribute over genomes of proteobacteria and actinobacteria. However, the biosynthetic gene clusters are cryptic in most cases. Therefore, the combination of genome mining and heterologous production is efficient method for the production of lasso peptides. To utilize lasso peptide as fine chemical, there have been several attempts to add new function to lasso peptide by genetic engineering. Currently, a more efficient lasso peptide production system is being developed to harness cryptic biosynthetic gene clusters of lasso peptide. In this review, the overview of lasso peptide study is discussed.
Asunto(s)
Familia de Multigenes , Péptidos , Actinobacteria/genética , Bacterias/genética , Péptido Hidrolasas/genética , Péptidos/química , ProteobacteriaRESUMEN
Microviridins are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) that have been isolated from a wide variety of cyanobacterial strains. There are similar gene clusters of RiPPs distributed in the genomes of bacteria belonging to the phyla Proteobacteria and Bacteroidetes. A cryptic gene cluster for the production of microviridin-type peptide was found in the genome of the marine γ-Proteobacterium Grimontia marina. Heterologous production of new microviridin-type peptide named grimoviridin was accomplished in Escherichia coli using the biosynthetic gene cluster of G. marina. The structure of grimoviridin was determined by analysis of MS and NMR data. Grimoviridin contained one isopeptide and two ester bonds, which had exactly the same bridging pattern as other microviridin-type peptides. The absolute stereochemistries of constituent amino acids were determined to be all L-forms by modified Marfey's method. Grimoviridin showed potent inhibitory activity against trypsin with an IC50 value of 238 nM. This is the first report of heterologous production of microviridin-type peptide using a biosynthetic gene cluster from a Proteobacterium. Key points ⢠Heterologous production afforded new microviridin-type peptide named grimoviridin. ⢠This is the first report of microviridin-type peptide from proteobacterial origin. ⢠Grimoviridin showed potent inhibitory activity against trypsin.